**4. Genetic engineering of glycine betaine (GB) synthesis improves drought tolerance in sugarcane**

Glycine betaine (*N,N,N*-trimethyl glycine) is an amphoteric small organic compound, highly soluble and do not interfere with cellular metabolism even at high concentration. The molecular character of GB can interact with macromolecule such as enzyme, protein complexes, and cell membrane when cell is exposed to stress condition. Glycine betaine stabilizes the structure and activity of enzymes and proteins, and maintains integrity of membrane against damage caused by environmental stresses [81]. This GB accumulates in a variety of plant species in response to water stresses for osmotic adjustment. Depending on the plant species, some plant species are accumulator of glycine betaine such as *Amaranthus*, sorghum, sugar beet and the other non-accumulators such as rice, sweet potato, and tobacco [5, 34]. Drought stress enhances accumulation of GB in the accumulator species for osmotic adjustment [82, 83]. Glycine betaine protects the plant cell by acting as an osmolyte, stabilizes protein and membrane cell, and maintains water balance during drought stress. It is widely accepted that the accumulation of GB plays an important role for the acclimatization of plant cell to drought stress. In many plants that do not accumulate GB, application of GB may help reduce adverse effects of the environment stress. The exogenous application of GB at 10 mM improved growth, leaf water content, and net photosynthesis, and increased growth and crop yield under environmental stress [84, 85]. However, consideration of economic and streamline useful application needs to be investigated. Determination of the GB concentration, timing, frequency of application, and a possibility of other disadvantage of exogenous GB application such as the risk of increasing pathogen attack should be well established [85]. Thus, genetic engineering for economically important crops such as rice, maize, and sugarcane that naturally are unable to accumulate GB will be an important target to improve.

selective agent [66]. Similar results reported that hygromycin is an effective selective marker for genetic transformation for monocot plants such as rice [67, 68], maize [69], and banana [70]. The herbicide Basta (*bar* gene) has been used as the selection marker for genetic transformation in rice [71], fescue—*Festuca arundinacea* [72], and oil palm [73]. However, limited reports have published the use of both *nptII*, *hptII*, and *bar* genes as effective selectable for sugarcane transformation. Using direct transformation with microprojectile bombardment, stable transformant was obtained after the selection of explant sugarcane callus on the media containing kanamycin and stepped increases in the antibiotic concentration allowed an active growing of callus, plantlets and completely inhibited untransformed callus [35]. When *Agrobacterium*mediated transformation was performed, similar results were observed using explant callus and *nptII* gene as the selectable marker. Regeneration transformant was successfully conducted by culturing the explant callus on the media containing 150 mg/L paromomycin sulfate [74]. However, the first successful report on *Agrobacterium*-mediated transformation for sugarcane used selectable marker *hptII* gene [57]. There are many reasons to elaborate the discrepancy between the effectiveness of selectable markers *nptII* or *hptII* genes, but hygromycin is much more effective than kanamycin for the selection of transformed cell and at low concentration, the hygromycin provides strong discrimination between transformed and nontransformed cell. Thus, hygromycin at the concentration of 25 mg/L is sufficient for routinely used *Agrobacterium*-

*Agrobacterium* strain and density frequently have an impact on the plant genetic transformation efficiency. There are many *Agrobacterium* strains used for genetic transformation in plant and, among of them, the LBA4404 *Agrobacterium* strain is widely used for genetic transformation. The LBA4404 has a higher transformation efficiency in several plants such as in tobacco [75], wheat [76], and herb of *Bacopa monnieri* [77]. However, GV3101 *Agrobacterium* strain has been reported with highest transformation rate than EHA105, AGL1, and MP90 *Agrobacterium* strains in tomato [78]. In addition, concentration of *Agrobacterium* and wounding explants are also considered as the factors influencing transgene expression in loblolly pine [79]. Concentration of *Agrobacterium* at OD<sup>600</sup> = 0.5 improved the efficiency of transformation in cotton [80], whereas higher concentration will result in *Agrobacterium* overgrowth and difficulty to eliminate after co-cultivation. In sugarcane transformation system using explant *in vitro* shoot, GV3101 *Agrobacterium* concentration at 0.5 OD600 and wounding explants can be

**4. Genetic engineering of glycine betaine (GB) synthesis improves** 

Glycine betaine (*N,N,N*-trimethyl glycine) is an amphoteric small organic compound, highly soluble and do not interfere with cellular metabolism even at high concentration. The molecular character of GB can interact with macromolecule such as enzyme, protein complexes, and cell membrane when cell is exposed to stress condition. Glycine betaine stabilizes the structure and activity of enzymes and proteins, and maintains integrity of membrane against damage caused by environmental stresses [81]. This GB accumulates in a variety of plant species in response to water stresses for osmotic adjustment. Depending on the plant species,

mediated transformation for sugarcane.

150 Sugarcane - Technology and Research

used for routinely transformation.

**drought tolerance in sugarcane**

Glycine betaine is an osmoprotectant found in wide range of microorganisms, plants, and animals that are synthesized under various environmental stresses [5]. Glycine betaine is mainly synthesized from choline as the substrate through two-step reactions, dehydrogenation of choline, and oxygenation of betaine aldehyde (**Figure 3**). In higher plants, choline is converted by choline monoxygenase (CMO) to betaine aldehyde, and then converted into glycine betaine by betaine aldehyde dehydrogenase (BADH) to GB [34, 86]. In microorganism and mammalian cells, GB is also synthesized by two-step pathway, but choline is converted to betaine aldehyde by choline dehydrogenase (CDH) and not by CMO, and then to GB by same BADH activity [34, 87]. In contrast, a single step-reaction catalyzed by choline oxidase (COD) for synthesis of GB is found in some microorganism such as *Arthrobacter globiformis* and *Arthrobacter panescens* [88] . In addition, a distinct substrate for GB synthesis is found in two halophytic microorganism *Actinopolyspora halophila* and *Ectothiorhodospira halochloris*. The GB is synthesized from substrate glycine by glycine methylation pathway [89]. The increasing knowledge of physiological pathway for GB biosynthesis as well as genomic engineering technology allow to create transgenic plants that are properly tolerant to drought stress by engineering of glycine betaine biosynthesis.

The gene involved in the biochemical pathway can be used either to increase or diminish metabolite product by overexpressing or silencing the gene responsible for the metabolism. In the case of metabolite engineering of GB, the enzymes involved in the biochemical pathway have been focused as a potential target to engineer the content in the non-accumulator plants. For that reason, the genes encoding for the enzymes involved in pathway of GB biosynthesis have been cloned from various microorganisms and plants that accumulate GB. In microorganism, gene encoding CDH (*bet*A) and BADH (*bet*B) have been isolated from *Escherichia coli* [90, 91] and from salt-tolerant bacteria *Halomonas elongata* [92], whereas gene encoding COD or COX was cloned from soil-living bacteria *Arthrobacter panescens* and *Arthrobacter globiformis* [93, 94]. In higher plants, limited number of genes encoding CMO in combination with BADH has been cloned from spinach [95], sugar beet, and amaranth [96]. Genes responsible for GB synthesis from microorganism have become a major target in the genetic engineering of water stress-tolerant in plants that are unable to accumulate GB, such as tomato (*Solanum lycopersicum*), potato (*Solanum tuberosum*), rice (*Oryza sativa*),

can enhance GB accumulation in transgenic plants. The GB accumulation that depend on the type of transgene and that of microorganism-derived gene for the pathway of GB synthesis is more potential, and constructing targeted gene under strong promoter is preferable. Although the GB was accumulated at low level, the transgenic plants expressing COD or COX showed

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 153

Plant tolerance environmental stress should be achieved by genetic engineering of BADH since the enzyme acts oxidizing betaine aldehyde into GB. The betaine aldehyde is a toxic compound and should not be accumulated in the cell. Transgenic carrot (*Daucus carota*) expressing BADH in the chloroplast grew well in the presence of NaCl up to 400 mM, whereas control non-transgenic plant showed severe growth retardation [105]. BADH enzyme activity, GB contents, and growth rate were enhanced under salt stress 100 mM NaCl in transgenic carrot compared to the control non-transgenic. Expression of cDNA for BADH from GB accumulator of spinach and sugar beet targeted in the chloroplast of transgenic tobacco increased the level of BADH activity, but failed to accumulate GB in the absence exogenous supply of betaine aldehyde [106]. Similar result was reported in transgenic tobacco transformed with cDNA for BADH from barley [107]. These results suggest that the increase in expression of BADH alone is not sufficient for the increase of GB in transgenic plant and that BADH might

Microorganism CDH is an useful enzyme for enhancing accumulation of GB into new species because the enzyme is able to catalyze in two reactions such as oxidation of choline to betaine aldehyde and also converting betaine aldehyde into GB [90, 109]. The purified recombinant CDH from *Halomonas elongata* showed similar subtract specificity with either choline or betaine aldehyde as the substrate [92]. Although gene encoding for CDH (*bet*A) has been poorly used for genetic engineering, increasing GB content and enhancing salt and drought tolerance have been proven in transgenic plants. Introducing *bet*A gene isolated from *E. coli* resulted in elevation of CDH activity and created salt tolerance phenotype of transgenic tobacco as well as an increase in the biomass dry weight [110]. The measurement of CDH activity in transgenic tobacco plants showed considerably higher CDH activity around 4.5–6 fold when compared with the wild type. In parallel with increasing CDH activity, the growth of transgenic tobacco was enhanced by salt stress at concentration 200–300 Mm NaCl. In addition, the level of endogenous GB was found to correlate with the degree of salt tolerance in maize lines and that the gene conferring GB plays a key role in osmotic adjustment [111]. Therefore, maize that transformed with the *bet*A gene from *E. coli* accumulated higher level of GB and more tolerant to drought stress than wild-type non-transgenic plants [98]. The measurement of CDH activities in transgenic maize showed increasing the activities 3-4 fold compared with non-transgenic maize and resulted in greater GB concentration compared with non-transgenic maize. The GB concentration was only 1.2 umol/g FW (fresh weight), but increased significantly up to 2.6–4.0 umol/g FW in the leaves of transgenic maize. Observation of total soluble sugar, free amino acids, and osmotic potential in the leaves of transgenic maize were not significantly increased, but photosystem II and net photosynthesis of transgenic maize were more stable than that in the control non-transgenic maize. These indicate that enhanced GB content has a beneficial effect on osmotic adjustment in condition of drought stress and protect from the damage due to dehydration. The most importantly, the grain yield of transgenic

enhanced environmental-stress tolerance and had better growth [34, 87].

possibly participate in other metabolite pathways [108].

**Figure 3.** The biosynthesis pathway of glycine betaine (GB) in microorganism and plant cells. Choline is oxidized to GB by two enzymes, choline monoxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) in plant cells. Depending on the species of microorganism, they operate two distinct pathways. In *Escherichia coli*, choline is oxidized to GB by choline dehydrogenase (CDH) and BADH, but in *Arthrobacter globiformis* choline is converted to GB by single enzyme of choline oxidase (COD).

and maize (*Zea mays*) [97, 98]. The strategy to enhance glycine betaine contents in transgenic plant can be achieved by introduction of the relevant gene under transcriptional control of strong DNA promoter to ensure high-level expression. The genetic engineering of drought-tolerant sugarcane, an important crop for sugar production in tropical areas, with overexpression of the *bet*A gene encoding for CDH from *E. coli* under control of 35S CaMV promoter will be discussed.

Genetic engineering of GB synthesis in plants has focused only with individual gene involved in the pathway of GB biosynthesis. The gene encoding COD from *Arthrobacter globiformis* has been overexpressed in chloroplast of *Arabidopsis* and accumulated in low level of GB [99, 100]. Similar results were obtained with low accumulation level of GB, when the COD was overexpressed in the chloroplast of transgenic rice [101] and COX was expressed in the chloroplast of three diverse species, *Arabidopsis*, *Brassica napus*, and *Nicotiana tabacum* (tabacco) [102]. In addition, the expression of CMO which is catalyzed oxidation of choline in plant cell, accumulated very low GB content in the chloroplast of transgenic tobacco [103] and transgenic rice [104]. The low level of GB in the transgenic plant due to limitation of choline contents in the site targeted synthesis of GB, where the synthesis of choline occurred in cytosol. When COD is targeted to express in the cytosol, the synthesis of GB was 3–5 fold greater when the enzyme was targeted to chloroplast [34]. Substantial increase of GB level was obtained when the transgenic plants expressing COX in their chloroplast feed with choline [102] and in transgenic tobacco expressing chloroplast CMO were supplied with choline or phosphocholine [103]. The GB accumulation in transgenic plants is affected by choline availability, type of transgene, and promoter type [87]. Availability of choline as the substrate for GB synthesis either exogenous or endogenous supply can enhance GB accumulation in transgenic plants. The GB accumulation that depend on the type of transgene and that of microorganism-derived gene for the pathway of GB synthesis is more potential, and constructing targeted gene under strong promoter is preferable. Although the GB was accumulated at low level, the transgenic plants expressing COD or COX showed enhanced environmental-stress tolerance and had better growth [34, 87].

Plant tolerance environmental stress should be achieved by genetic engineering of BADH since the enzyme acts oxidizing betaine aldehyde into GB. The betaine aldehyde is a toxic compound and should not be accumulated in the cell. Transgenic carrot (*Daucus carota*) expressing BADH in the chloroplast grew well in the presence of NaCl up to 400 mM, whereas control non-transgenic plant showed severe growth retardation [105]. BADH enzyme activity, GB contents, and growth rate were enhanced under salt stress 100 mM NaCl in transgenic carrot compared to the control non-transgenic. Expression of cDNA for BADH from GB accumulator of spinach and sugar beet targeted in the chloroplast of transgenic tobacco increased the level of BADH activity, but failed to accumulate GB in the absence exogenous supply of betaine aldehyde [106]. Similar result was reported in transgenic tobacco transformed with cDNA for BADH from barley [107]. These results suggest that the increase in expression of BADH alone is not sufficient for the increase of GB in transgenic plant and that BADH might possibly participate in other metabolite pathways [108].

Microorganism CDH is an useful enzyme for enhancing accumulation of GB into new species because the enzyme is able to catalyze in two reactions such as oxidation of choline to betaine aldehyde and also converting betaine aldehyde into GB [90, 109]. The purified recombinant CDH from *Halomonas elongata* showed similar subtract specificity with either choline or betaine aldehyde as the substrate [92]. Although gene encoding for CDH (*bet*A) has been poorly used for genetic engineering, increasing GB content and enhancing salt and drought tolerance have been proven in transgenic plants. Introducing *bet*A gene isolated from *E. coli* resulted in elevation of CDH activity and created salt tolerance phenotype of transgenic tobacco as well as an increase in the biomass dry weight [110]. The measurement of CDH activity in transgenic tobacco plants showed considerably higher CDH activity around 4.5–6 fold when compared with the wild type. In parallel with increasing CDH activity, the growth of transgenic tobacco was enhanced by salt stress at concentration 200–300 Mm NaCl. In addition, the level of endogenous GB was found to correlate with the degree of salt tolerance in maize lines and that the gene conferring GB plays a key role in osmotic adjustment [111]. Therefore, maize that transformed with the *bet*A gene from *E. coli* accumulated higher level of GB and more tolerant to drought stress than wild-type non-transgenic plants [98]. The measurement of CDH activities in transgenic maize showed increasing the activities 3-4 fold compared with non-transgenic maize and resulted in greater GB concentration compared with non-transgenic maize. The GB concentration was only 1.2 umol/g FW (fresh weight), but increased significantly up to 2.6–4.0 umol/g FW in the leaves of transgenic maize. Observation of total soluble sugar, free amino acids, and osmotic potential in the leaves of transgenic maize were not significantly increased, but photosystem II and net photosynthesis of transgenic maize were more stable than that in the control non-transgenic maize. These indicate that enhanced GB content has a beneficial effect on osmotic adjustment in condition of drought stress and protect from the damage due to dehydration. The most importantly, the grain yield of transgenic

and maize (*Zea mays*) [97, 98]. The strategy to enhance glycine betaine contents in transgenic plant can be achieved by introduction of the relevant gene under transcriptional control of strong DNA promoter to ensure high-level expression. The genetic engineering of drought-tolerant sugarcane, an important crop for sugar production in tropical areas, with overexpression of the *bet*A gene

**Figure 3.** The biosynthesis pathway of glycine betaine (GB) in microorganism and plant cells. Choline is oxidized to GB by two enzymes, choline monoxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) in plant cells. Depending on the species of microorganism, they operate two distinct pathways. In *Escherichia coli*, choline is oxidized to GB by choline dehydrogenase (CDH) and BADH, but in *Arthrobacter globiformis* choline is converted to GB by single enzyme

Genetic engineering of GB synthesis in plants has focused only with individual gene involved in the pathway of GB biosynthesis. The gene encoding COD from *Arthrobacter globiformis* has been overexpressed in chloroplast of *Arabidopsis* and accumulated in low level of GB [99, 100]. Similar results were obtained with low accumulation level of GB, when the COD was overexpressed in the chloroplast of transgenic rice [101] and COX was expressed in the chloroplast of three diverse species, *Arabidopsis*, *Brassica napus*, and *Nicotiana tabacum* (tabacco) [102]. In addition, the expression of CMO which is catalyzed oxidation of choline in plant cell, accumulated very low GB content in the chloroplast of transgenic tobacco [103] and transgenic rice [104]. The low level of GB in the transgenic plant due to limitation of choline contents in the site targeted synthesis of GB, where the synthesis of choline occurred in cytosol. When COD is targeted to express in the cytosol, the synthesis of GB was 3–5 fold greater when the enzyme was targeted to chloroplast [34]. Substantial increase of GB level was obtained when the transgenic plants expressing COX in their chloroplast feed with choline [102] and in transgenic tobacco expressing chloroplast CMO were supplied with choline or phosphocholine [103]. The GB accumulation in transgenic plants is affected by choline availability, type of transgene, and promoter type [87]. Availability of choline as the substrate for GB synthesis either exogenous or endogenous supply

encoding for CDH from *E. coli* under control of 35S CaMV promoter will be discussed.

of choline oxidase (COD).

152 Sugarcane - Technology and Research

maize, overexpressing *bet*A gene from *E. coli*, was significantly higher than that of the control non-transgenic after drought treatments [98]. Similar result is also reported that transgenic cotton (*Gossypium hirsutum* L.) expressing *bet*A gene from *E. coli* enhanced GB accumulation and drought tolerance [112]. The GB concentration in the leaves of transgenic cotton was higher compared to wild-type plants in non-stress condition and the GB concentration was much elevated after drought stress condition reached at 282.3–308.4 umol/g dry weight or around 2.3–2.6 fold compared with wild-type plants. The measurement of osmotic pressure showed that the osmotic adjustment was higher in transgenic than in the wild type plant, and the higher osmotic adjustment was significantly correlated with the GB concentration in drought stress condition. The results indicate that GB plays a more important role in osmotic adjustment and maintains the membrane stability than that in slightly increased of soluble sugar and amino acid contents in transgenic cotton. Moreover, after 4 days drought stress treatments, the wild-type cotton showed starting to wilt and decreased leaf relative water contents, whereas wilting symptom did not observe and the leaf relative water content remained higher until 10 days of the drought treatments in transgenic cotton. As observed in the transgenic maize, the transgenic cotton seed yield is significantly greater than in wild-type non-transgenic cotton under drought stress [112]. All together, these studies imply that engineering of GB content using *bet*A gene for CDH from *E. coli* enhances GB content that acts not only as an osmoprotectant, but also stabilizes the structure and activities of protein-enzymes, and maintains the integrity of membrane against damage caused by the drought stress, which in turn increase growth and productivity of the transgenic plants.

with Inertsil ODS-3 column. The GB content highly elevated in the leaves of transgenic sugarcane ranged 182–880 ppm, but almost not detected in the control non-transgenic sugarcane after drought condition. The increasing of GB contents sugarcane enhanced drought-tolerance of transgenic sugarcane. Observation of plant morphology during exposure to drought stress by stop watering showed that non-transgenic sugarcane started to wilt at 8 days and permanently wilt at 28 days after drought stress. However, the transgenic sugarcane still vigorously growth at 8 days and start to wilt at 12 days, and then permanently wilt after more than 30 days of drought stress treatments (**Figure 4A, B**). Moreover, expressing gene *betA* also induced salt-tolerance of the transgenic sugarcane. When cultured in media containing 200 mM NaCl for 3 weeks, the drought-tolerance sugarcane showed stay-green, but the wilt-type leaves were yellowish and partly dried. Interestingly, observation of the root profile of the transgenic sugarcane showed a wider and longer root system compared to the wild-type sugarcane, but there was no change in the appearance of the shoot morphology (**Figure 4C**). The improved root system has a good water absorption system to extract limited water availability from deep soil and this is a criterion of drought-tolerance sugarcane [114, 115]. These results imply that the enhanced GB contents in transgenic sugarcane provides an osmoprotectant, stabilizes the structure of macromolecule, maintains the integrity and proper function of the cell membranes, and helps the sugarcane acclimate to drought-

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 155

To investigate the growth and productivity of transgenic sugarcane under water limited condition, the sugarcane was grown in non-irrigated dry land of experiment station. Cultivation of the transgenic sugarcane was carried out under confined and limited field trial system according to the regulation for assessment of genetically modified organisms (GMO). Comparison of the drought-tolerance transgenic sugarcane with the wild-type showed almost no difference in the germination of lateral buds and the initial growth rate.

**Figure 4.** Growth performance of drought-tolerant sugarcane overexpressed *betA* gene in greenhouse and in nonirrigated experiment field station using confined trials system. Two-months old NXI-4T (transgenic drought-tolerant) and BL (non-transgenic) sugarcane cultivars were grown in greenhouse and treated with drought stress by stop watering. The transgenic drought-tolerant NXI-4T sugarcane stay green (left) and non-transgenic BL sugarcane cultivars (right) started wilting (A) after 8 days drought stress, then BL sugarcane being permanently wilting and dried after 28 days without watering (B). Wider and longer root profile of NXI-4T (right) is compared to BL (left) sugarcane (C). Normally growth internode of NXI-4T (D) and retarded internode of control BL sugarcane cultivars (E). Growth performance of nine-months old drought-tolerant sugarcane (F). The figures were provided by Dr. Nurmalasari of PT. Perkebunan

and salt-stress condition.

Nusantara XI, Surabaya, Indonesia.

Several experiments on the effect of addition exogenous GB have been reported to improve sprouting sugarcane bud under heat and chilling stresses [113]. However, there is almost no report concerning accumulation of GB content in sugarcane. Therefore, enhancing GB synthesis with genetic engineering is considered to be a potential method for improving drought stress tolerance in sugarcane. The drought-tolerance transgenic sugarcane has been developed by introduction of *betA* gene encoding for CDH from *Rhizobium meliloti* (Australian Patent Office, Patent No. 737600 – Inventor(s); Naoki Katsurada, Tsushi Hayakawa, Haruhumi Miwa). The *bet*A gene was constructed in binary vector under the control of strong promoter CaMV35S by Ajinomoto Co., Inc., Japan and used for sugarcane transformation. The *Agrobacterium*mediated transformation was conducted using explant from BL sugarcane cultivars by the state-run sugarcane producer PT Perkebunan Nusantara XI Indonesia in collaboration with Ajinomoto company and University of Jember. After screening of transformed plantlet in selection media containing hygromycin antibiotic, the resulted plantlet was acclimated in green house and used for analysis. The characterization of the transgenic sugarcane was carried out by analysis of the plants grown in greenhouse experiments.

Genomic analysis by PCR (polymerase chain reaction) and Southern Blot confirmed the presence of stable insertion of *bet*A in the genome of the transgenic sugarcane. As expected, the transgenic sugarcane lines appeared to contain a low-copy insertion of *bet*A gene, whereas non-transgenic plant almost had no DNA hybridization. The stable integration of *bet*A gene was confirmed by PCR analysis in third generation of transgenic sugarcane after vegetative propagation. Although the expressions of gene *bet*A at transcriptional and translational levels were not examined, GB contents in the transgenic sugarcane were detected using HPLC with Inertsil ODS-3 column. The GB content highly elevated in the leaves of transgenic sugarcane ranged 182–880 ppm, but almost not detected in the control non-transgenic sugarcane after drought condition. The increasing of GB contents sugarcane enhanced drought-tolerance of transgenic sugarcane. Observation of plant morphology during exposure to drought stress by stop watering showed that non-transgenic sugarcane started to wilt at 8 days and permanently wilt at 28 days after drought stress. However, the transgenic sugarcane still vigorously growth at 8 days and start to wilt at 12 days, and then permanently wilt after more than 30 days of drought stress treatments (**Figure 4A, B**). Moreover, expressing gene *betA* also induced salt-tolerance of the transgenic sugarcane. When cultured in media containing 200 mM NaCl for 3 weeks, the drought-tolerance sugarcane showed stay-green, but the wilt-type leaves were yellowish and partly dried. Interestingly, observation of the root profile of the transgenic sugarcane showed a wider and longer root system compared to the wild-type sugarcane, but there was no change in the appearance of the shoot morphology (**Figure 4C**). The improved root system has a good water absorption system to extract limited water availability from deep soil and this is a criterion of drought-tolerance sugarcane [114, 115]. These results imply that the enhanced GB contents in transgenic sugarcane provides an osmoprotectant, stabilizes the structure of macromolecule, maintains the integrity and proper function of the cell membranes, and helps the sugarcane acclimate to droughtand salt-stress condition.

maize, overexpressing *bet*A gene from *E. coli*, was significantly higher than that of the control non-transgenic after drought treatments [98]. Similar result is also reported that transgenic cotton (*Gossypium hirsutum* L.) expressing *bet*A gene from *E. coli* enhanced GB accumulation and drought tolerance [112]. The GB concentration in the leaves of transgenic cotton was higher compared to wild-type plants in non-stress condition and the GB concentration was much elevated after drought stress condition reached at 282.3–308.4 umol/g dry weight or around 2.3–2.6 fold compared with wild-type plants. The measurement of osmotic pressure showed that the osmotic adjustment was higher in transgenic than in the wild type plant, and the higher osmotic adjustment was significantly correlated with the GB concentration in drought stress condition. The results indicate that GB plays a more important role in osmotic adjustment and maintains the membrane stability than that in slightly increased of soluble sugar and amino acid contents in transgenic cotton. Moreover, after 4 days drought stress treatments, the wild-type cotton showed starting to wilt and decreased leaf relative water contents, whereas wilting symptom did not observe and the leaf relative water content remained higher until 10 days of the drought treatments in transgenic cotton. As observed in the transgenic maize, the transgenic cotton seed yield is significantly greater than in wild-type non-transgenic cotton under drought stress [112]. All together, these studies imply that engineering of GB content using *bet*A gene for CDH from *E. coli* enhances GB content that acts not only as an osmoprotectant, but also stabilizes the structure and activities of protein-enzymes, and maintains the integrity of membrane against damage caused by the drought stress, which in turn increase growth and productiv-

Several experiments on the effect of addition exogenous GB have been reported to improve sprouting sugarcane bud under heat and chilling stresses [113]. However, there is almost no report concerning accumulation of GB content in sugarcane. Therefore, enhancing GB synthesis with genetic engineering is considered to be a potential method for improving drought stress tolerance in sugarcane. The drought-tolerance transgenic sugarcane has been developed by introduction of *betA* gene encoding for CDH from *Rhizobium meliloti* (Australian Patent Office, Patent No. 737600 – Inventor(s); Naoki Katsurada, Tsushi Hayakawa, Haruhumi Miwa). The *bet*A gene was constructed in binary vector under the control of strong promoter CaMV35S by Ajinomoto Co., Inc., Japan and used for sugarcane transformation. The *Agrobacterium*mediated transformation was conducted using explant from BL sugarcane cultivars by the state-run sugarcane producer PT Perkebunan Nusantara XI Indonesia in collaboration with Ajinomoto company and University of Jember. After screening of transformed plantlet in selection media containing hygromycin antibiotic, the resulted plantlet was acclimated in green house and used for analysis. The characterization of the transgenic sugarcane was car-

Genomic analysis by PCR (polymerase chain reaction) and Southern Blot confirmed the presence of stable insertion of *bet*A in the genome of the transgenic sugarcane. As expected, the transgenic sugarcane lines appeared to contain a low-copy insertion of *bet*A gene, whereas non-transgenic plant almost had no DNA hybridization. The stable integration of *bet*A gene was confirmed by PCR analysis in third generation of transgenic sugarcane after vegetative propagation. Although the expressions of gene *bet*A at transcriptional and translational levels were not examined, GB contents in the transgenic sugarcane were detected using HPLC

ried out by analysis of the plants grown in greenhouse experiments.

ity of the transgenic plants.

154 Sugarcane - Technology and Research

To investigate the growth and productivity of transgenic sugarcane under water limited condition, the sugarcane was grown in non-irrigated dry land of experiment station. Cultivation of the transgenic sugarcane was carried out under confined and limited field trial system according to the regulation for assessment of genetically modified organisms (GMO). Comparison of the drought-tolerance transgenic sugarcane with the wild-type showed almost no difference in the germination of lateral buds and the initial growth rate.

**Figure 4.** Growth performance of drought-tolerant sugarcane overexpressed *betA* gene in greenhouse and in nonirrigated experiment field station using confined trials system. Two-months old NXI-4T (transgenic drought-tolerant) and BL (non-transgenic) sugarcane cultivars were grown in greenhouse and treated with drought stress by stop watering. The transgenic drought-tolerant NXI-4T sugarcane stay green (left) and non-transgenic BL sugarcane cultivars (right) started wilting (A) after 8 days drought stress, then BL sugarcane being permanently wilting and dried after 28 days without watering (B). Wider and longer root profile of NXI-4T (right) is compared to BL (left) sugarcane (C). Normally growth internode of NXI-4T (D) and retarded internode of control BL sugarcane cultivars (E). Growth performance of nine-months old drought-tolerant sugarcane (F). The figures were provided by Dr. Nurmalasari of PT. Perkebunan Nusantara XI, Surabaya, Indonesia.

However, with the start of dry season, non-transgenic sugarcane showed retardation and elongation of stem. The internode of non-transgenic sugarcane becomes shorten during the dry season, but not the internode of transgenic sugarcane (**Figure 4D, E**). The internode of transgenic sugarcane was normally elongated at the similar size as expected and was not affected by dry season. The measurement of sugarcane yield of cane stalk significantly increased compared with non-transgenic plants, although the sucrose content was not different. Similar results were observed that drought-tolerance sugarcane lines have higher productivity stalk height and stalk weight than the susceptible line [116, 117]. In conclusion, all together the results showed that the transgenic sugarcane expression *betA* gene is a drought-tolerant sugarcane and this sugarcane should be the first drought-tolerant sugarcane developed by biotechnology approach.

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The Cartagena Protocol on Biosafety (2000), which protects biological diversity from the potentially risk due to the use of transgenic plants, has been ratified by Indonesian government. Thus, for the commercialization of drought-tolerance sugarcane, biosafety assessment has been completed such as environmental safety, food, and feed safety. The environment safety assessment claimed that the drought-tolerance sugarcane has no effect on biodiversity, the occurrence of gene flow, and potentially to be an invasive crop. Bioinformatics BLASTP analysis suggested CDH protein encoded by *bet A* did not have similarity with allergen data base (NCBI Entrez) and potentially to be allergen. Further analysis using animal feeding experiment and simulation digestion system found that the drought-tolerance sugarcane did not potentially toxic or allergen. Based on the biosafety assessment, the drought-tolerance of sugarcane has been approved by the National Genetically Modified Product Biosafety Commission for commercial cultivation in the state-run sugarcane producer PT. Perkebunan Nusantara XI [118]. The company claimed that the drought-tolerance sugarcane produces 10–30% higher sugar productivity under dry land than in conventional parental lines.
